The long-term goal of this project is to use Homology Directed Repair (HDR) induced by meganucleases to correct mutations in the mouse &#946;-glucuronidase gene and the human &#946;-globin gene in induced Pluripotent Stem (iPS) cells. HDR is achieved by creating a double stranded break (DSB) near a mutation of interest with a site-specific meganuclease, followed by repair with a wild-type [unreadable]correcting DNA[unreadable]. Two classes of meganucleases (zinc-finger and homing) are currently being evaluated for their ability to induce site-specific DSBs, but these enzyme classes have not been directly compared for their ability to induce repair. Although meganuclease-initiated HDR is thought to be more efficient than traditional homologous recombination approaches, these enzymes carry a risk of [unreadable]off-target[unreadable] DNA cleavage events and the creation of additional mutations. To address these issues, we propose the following Specific Aims: Specific Aim 1: We will use homology directed repair to correct the point mutation that causes murine Mucopolysaccaridosis Type VII (MPS VII). We have already obtained 6 different homing meganucleases that specifically cleave at or near the point mutation that inactivates the mouse &#946;-glucuronidase (Gusb) gene, causing MPS VII. ES cells, iPS cells, and bone marrow cells derived from Gusb deficient mice will be transfected with cDNAs encoding each of the meganucleases, and quantitatively assessed for restoration of enzyme activity using a highly sensitive flow-based assay for &#946;-glucuronidase activity. Corrected cells will be transferred to Gusb deficient mice to determine whether they can mitigate the disease phenotype. Specific Aim 2: We will use homology directed repair to correct the point mutation that causes sickle cell anemia. We have already obtained 6 different homing meganucleases and 1 zinc-finger meganuclease that cleave within the human &#946;-globin gene. We will transfect the cDNAs encoding these enzymes into iPS cell lines derived from sickle cell patients, and assess HDR efficiency for each. Hematopoietic progenitors obtained from the corrected iPS cells will be tested for their ability to produce functional red blood cells. Specific Aim 3: We will completely sequence the genomes of iPS cell lines that have undergone homology directed repair initiated by meganucleases. We will use [unreadable]next generation[unreadable] DNA sequencing to produce 8x haploid coverage of iPS cells derived from Gusb deficient mice, and gene-corrected subclones from these lines. Similarly, we will generate 25x haploid coverage of the primary fibroblasts, iPS lines, and gene-corrected iPS lines from each of several sickle cell anemia patients. We will define the mutations that were generated during the creation of the iPS lines, and subsequent correction of the Gusb or &#946;-globin mutations. These studies will be essential for understanding the potential risks of using gene-corrected iPS cells for human therapeutic trials.